Detection And Genetic Characterization Of Enterovirus71in Children With Severe Hand, Foot And Mouth Disease

ObjectiveTo clarify the etiological characteristic of HFMD in98children hospitalized in Tianjin Children’s Hospital from July to September in2012, understand the value of different clinical samples and methods for the early stage diagnosis of enterovirus infection, and explore the genetic characteristics of EV71by amplifing its full-length VP1gene.Method79throat swabs,86stool samples and23cerebrospinal fluid samples were collected from98hospitalized children with severe hand, foot and mouth disease. Among these cases,67children have both throat swabs and stool samples. Clinical data were recorded for every children. Total RNA was extracted from those samples, then three channel one-step real-time fluorescent RT-PCR was performed to detect the RNAs of general human enterovirus (EV), enterovirus71(EV71) and coxsackievirus A16(CA16). At the same time, ELISA was carried out to detect the IgM of EV71in the serum of patients. Finally, the full-length VP1gene of EV71was amplified from the samples positive for EV71nucleotide. Blast and phylogenetic analysis was performed.ResultsAmong the98severe cases,60.2%(59/98) were positive for EV71,10.2%(10/98) were positive for CA16and16.3%(16/98) were positive for other enterovirus. The positive rate of EV is82.7%(81/98). Moreover, four children were positive for both EV71and CA16. The male/female ratio in severe HFMD was about2:1. Children under five years old account for95.9%of all the severe cases. But75.5%were between6months and2years. The virus positive rate of children under2years old was85.7%, which was higher than that of children older than2years old, with no statistical significance. Stool samples had a higher virus positive rate (82.6%) than throat swabs (53.2%). No positive nucleic acid were detected in23 cerebrospinal fluid specimens. According to the results of ELISA,70.4%of the children in our study had IgM antibodies for EV71, which is higher than the results of fluorescent RT-PCR (60.2%). Phylogenetic analysis of the VP1gene from16EV71strains suggests that strains of EV71causing severe HFMD belong to C4a in Tianjin,2012.Conclusion1. EV71was still the major pathogen for the prevalence of severe HFMD in Tianjin area in2012, following was other enterovirus, the rate of CA16had declined.2. Children under five years old were susceptible to severe HFMD, especially those who were under2years old. It should be alert that young children, are more likely to develop severe HFMD. However, there is no significant gender and age differences for the detection of enterovirus.3. Stool samples were more suitable to etiologic detection, for it had a higher virus positive rate than throat swabs. Rare positive results were seen in cerebrospinal fluid samples.4. The detection of IgM anti-EV71by ELISA affords a reliable, convenient, and prompt diagnosis of EV71infection, and can meet the demand of preventing and controlling EV71infection for grassroots medical institutions.5. Strains of EV71causing severe HFMD belong to C4a in Tianjin,2012.